1. Field of the Invention
The invention relates to a method of improving protein production by cultured animal cells. More specifically, the invention relates to a method for controlling lactic acid production by cultured animal cells (preferably mammalian cells) to low levels in a fed-batch cell culture. In some embodiments, the invention provides methods of maintaining lactate production by cultured cells at low levels through the use of glucose delivery systems that do not rely on the sampling of cultures at regular intervals. In particular, the invention relates to culturing animal cells under conditions wherein glucose is fed to or into the culture in a restricted manner, e.g., at a rate that is a function of an expected or a premodeled rate of glucose consumption by the animal cells when they are exposed to a medium containing a high level of glucose. In association with this restricted feeding, the production by cultured cells of lactic acid is controlled to a low level during culture. As an end result, recombinant protein production from the cultured cells is increased, for example, in order to facilitate commercial-scale production.
2. Related Background Art
A large proportion of biotechnology products, whether commercially available or only in development, are protein therapeutics. Furthermore, the cellular machinery of an animal cell (versus a bacterial cell) generally is required in order for many forms of protein therapeutics (such as glycosylated proteins or hybridoma-produced monoclonal antibodies (MAbs)) to be produced. Consequently, there is an increasing demand for production of these proteins in animal cell cultures.
As compared to bacterial cell cultures, however, animal cell cultures have lower production rates and typically generate lower production yields. Maintaining glucose concentrations in cell culture media at low concentrations (e.g., between 0.02 and 1.0 g/L (e.g., between 0.11 and 5.5 mM)) and culturing cells in a production phase at an osmolality of about 400 to 600 mOsm has been found to increase production of recombinant proteins by animal cell cultures, particularly after an initial culturing at an osmolality of about 280 to 330 mOsm (U.S. Pat. No. 5,856,179; each U.S. patent cited in this document is incorporated by reference in its entirety) and wherein culturing in all phases is also at a selected glutamine concentration (preferably between about 0.2 to about 2 mM; U.S. Pat. No. 6,180,401).
Some of this increase in recombinant protein production may result from a reduction in lactate production that occurs when glucose concentrations in culture media are maintained at low levels. Lactate is known to be a strong inhibitor of cell growth and protein production, and maintaining low glucose concentrations in culture media can result in low levels of lactate production (Glacken et al. (1986) Biotechnol. Bioeng. 28:1376-89; Kurokawa et al. (1994) Biotechnol. Bioeng. 44:95-103; U.S. Pat. No. 6,156,570). Consequently, depending on other culture conditions, maintaining glucose concentrations at low levels relative to cell concentration is one factor that can contribute to lower levels of lactate production, and thus to higher cell concentrations and increased production of recombinant proteins in animal cell cultures.
When cells are exposed to low glucose concentration in a medium, their metabolism is altered such that both glucose uptake rate and lactate production rate are lower as compared to cells maintained in fed-batch processes having media with high glucose levels at the start of the process (U.S. Pat. No. 6,156,570). Furthermore, the duration of the fed-batch culture can be extended. Consequently, both cell growth rate and protein production rate can be maintained for a longer period as compared to control fed-batch cultures in which cells are grown in media conducive to high levels of lactate production (e.g., media containing high glucose levels at the start of the culture period).
One way to control lactate production by cultured cells to low levels is through an invariant, constant-rate feeding of glucose in a fed-batch process (Ljunggren and Häggström (1994) Biotechnol. Bioeng. 44:808-18; Häggström et al. (1996) Annals N.Y. Acad. Sci. 782:40-52). Although this invariant, constant-rate feeding of glucose in a fed-batch process can help control lactic acid production by cultured cells to low levels, maximum cell concentrations, growth rates, cell viability levels, and protein production rates are not achieved, because this method of providing glucose typically results in glucose starvation as cell concentrations increase.
Another way to control lactate production by cultured cells to low levels is through the use of glucose delivery systems that rely on sampling cultures at regular intervals. Samples are taken from a culture at regular intervals, and, after the glucose concentration in samples is determined (e.g., through flow injection analysis, as by Male et al. (1997) Biotechnol. Bioeng. 55:497-504, or Siegwart et al. (1999) Biotechnol. Prog. 15:608-16; or through high pressure liquid chromatography, as by Kurokawa et al. (1994) Biotechnol. Bioeng. 44:95-103), measured amounts of glucose are added to the cultures in order to maintain glucose concentrations in media at a sustained low level relative to cell concentration. However, cells may adapt to low glucose concentration by, for example, increasing their ability to take in glucose, and thus produce excessive amounts of lactic acid despite the low glucose concentration.
Furthermore, the risk of microorganism contamination through such sampling-based feedback control methods is significant. Consequently, it is not surprising that the use of these methods for the commercial production of recombinant proteins in animal cell cultures has not proved feasible. The sampling-based feedback control methods for maintaining low levels of glucose concentration in cell culture media have been limited to research uses from the time the early article on such methods was published (Glacken et al., supra); the paper reports that glucose concentrations in culture media were determined by using an on-line autoanalyzer, wherein a glucose-containing sample was mixed with o-toluidine, and a colorimeter, through which the absorbance at 660 nm of the mixture was measured to determine glucose concentration.
For the foregoing reasons there is a need for alternative methods of controlling lactate production by cultured cells to low levels in culture media.